The research conducted during the first two years of the current grant period involved the determination of nucleotide sequences in specific regions of prokaryotic DNA molecules and the use of recombinant DNA techniques to study the sequence organization and expression of eukaryotic DNA molecules. The emphasis of this continuation grant proposal is the application of these results and techniques to the study of a variety of biologically interesting regions of eukaryotic DNA molecules. It is proposed to screen a bank of randomly cloned yeast DNA fragments in E. coli for those fragments which possess yeast replication origins. The selection procedure will utilize pulse-labeled DNA obtained from synchronized yeast cells as they are released from a block at the beginning of S phase in the cell cycle. The replication origin sites on the cloned yeast fragments will be characterized with respect to their location in or near known structural genes, their kinetic complexity, their actual nucleotide sequence and several other properties. Restriction enzyme fragments of yeast mitochondrial DNA have been cloned in E. coli and will be used as probes to determine if any mitochondrial transcription events are coordinated with cell cycle events. The cloned fragments will also be used to search for the specific replication origin(s) of yeast mitochondrial DNA - site(s) about which very little is known.